1. Technical Field
The field of this invention is assays involving cellular response to compounds of interest.
2. Background
There are a number of different situations where one is interested in the cellular response to a compound. In the screening of test compounds, the sensitivity of aberrant or normal cells to agents, or elucidating biological pathways, there is an interest in whether a compound will bind to a receptor in the cellular environment, the transduction of a signal from a membrane into a cellular compartment, or the response of the cell to the agent. While screening compounds solely for binding provides for rapid screening capabilities, the information content is limited due to the restricted nature of the assay.
Using cells as the target for the compounds has many advantages in allowing for a more natural environment for binding, where the receptor is in its natural environment and will be associated with the membrane and other proteins, which may complex with the receptor. Where the cell is viable, there is the potential for detecting the influence of the compound on the biological pathways of the cell, which in many situations may be essential for evaluating the potential of the compound. A viable cell gives an immediate indication of toxicity of the compound and depending on the circumstances, will allow for the determination of transcription, expression, ion channel activity, and the like. Where cells have been genetically engineered, there is the further opportunity to provide for a specific target for the transduced signal, where expression produces a detectable signal, such as green fluorescent protein or an enzyme, which acts on a substrate to provide a detectable signal. Such enzymes include xcex2-galactosidase, luciferase, etc.
In many instances, the amount of candidate compound and one or more reagents which is available is very small, being rare and/or expensive, so one wishes to have as small a reaction volume as possible. In addition, there are advantages to be able to identify the results with individual cells, so that a distribution of values is obtained, rather than a single value from a bulk medium or a value averaged over many cells.
One potential method for assaying the effect of compounds on cells is capillary electrokinesis. Concerns associated with the use of capillary electrokinesis are the maintenance of cellular viability, clumping and aggregation of cells, capillary wall adhesion, control of cellular movement and the effect of the medium on the binding of compounds to their respective receptors and transport across the cellular membrane. Particularly, as to viability, the saltconcentrations employed for cell media are drastically higher than are useful for electrokinesis. However, the advantages of being able to move small volumes of predetermined volume and concentration, permitting relatively high concentrations with small amounts of materials, detecting the effects on individual cells and being able to do numerous determinations simultaneously, makes the use of capillary electrokinesis an attractive opportunity.
3. Brief Description of Relevant Art
WO98/002321 describes a microfluidic apparatus for high throughput screening. U.S. Pat. No. 5,843,680 describes an apparatus for analyte detection. U.S. Pat. No. 5,260,192 describes an apparatus for screening cells. WO98/10267 and 98/43066 describe microflow systems for separating particles. U.S. Pat. No. 5,876,675 describes a microfluidic device for performing experiments including cell separation. The Zare group has published a number of papers concerning transport of analytes using capillary electrophoresis for cellular assays. See, for example, Jardemark, et al., Anal. Chem. 1998, 70, 2468-2474; Chiu, et al., ibid 1997, 69, 1801-1807; and Fishman et al., ibid, 1996, 68, 1181-1186.
Cellular operations are performed in a microfluidic device, employing a reservoir of cells, moving cells individually from the reservoir into a capillary channel by means of electrokinesis, contacting individual cells with one or more appropriate agents under conditions for performance of the operation and determining the results of the operation. Viable cells, particularly mammalian cells, may be employed in the process in a viability-maintaining environment or fixed cells may be used in media which are not restricted as to composition.